Maturation-dependent reduced responsiveness of intracellular free Ca2+ ions to repeated stimulation by N-methyl-d-aspartate in cultured rat cortical neurons

金沢大学大学院自然科学研究科分子作用学In contrast to other ionotropic glutamate receptors, N-methyl-d-aspartate (NMDA) receptor channels are rather stable after the simulation. Brief exposure to NMDA at 50 μM rapidly increased the fluorescence intensity for increased intracellular free Ca2+ levels in a reversible- and concentration-dependent manner in rat cortical neurons cultured for 3-15 days in vitro (DIV), while EC50 values were significantly decreased in proportion to cellular maturation from 3 to 15 DIV. Although a constant increase was persistently seen in the fluorescence throughout the sustained exposure to NMDA for 60 min irrespective of the cell maturation from 3 to 15 DIV, the second brief exposure for 5 min resulted in a less efficient increase in the fluorescence than that found after the first brief exposure for 5 min in a manner dependent on intervals between the two repetitive brief exposures. In vitro maturation significantly shortened the interval required for the reduced responsiveness to the second brief exposure, while in immature neurons prolonged intervals were required for the reduced responsiveness to the second brief exposure to NMDA. Moreover, brief exposure to NMDA led to a marked decrease in immunoreactivity to extracellular loop of NR1 subunit in cultured neurons not permeabilized in proportion to the time after washing. These results suggest that cellular maturation would facilitate the desensitization process to repeated stimulation by NMDA, without markedly affecting that to sustained stimulation, through a mechanism related to the decreased number of NMDA receptors expressed at cell surfaces in cultured rat cortical neurons. © 2006 Elsevier Ltd. All rights reserved

NMDAレセプターチャネルのメンブラントラフィック機構

金沢大学理工研究域グルタメイト(Glu)は、中枢神経系では興奮性神経情報伝達物質としての機能とともに、内在性興奮毒としての神経毒性を発揮するが、いずれの機能出現にも細胞膜上に発現する受容体を仲介するシグナルトランスダクションが関与すると理解される。Glu受容体の中でも特にNMDA受容体(NMDAR)の機能異常は、一過性脳虚血後の遅発性神経細胞死、およびアルツハイマー病やパーキンソン病、統合失調症など、種々の神経変性疾患の発症メカニズムに深く関与すると考えられている。そこで本研究は、NMDAシグナル伝達系の基礎的メカニズム解明を通じて、NMDAR機能異常に関連する神経変性疾患に対する新規治療法および予防法の探索を目的として行った。その結果、Ca^感受性蛍光指示薬を取り込ませたラット大脳皮質由来初代培養神経細胞に継続的なNMDA曝露を行っても持続的な蛍光強度の上昇が観察されるが、短期間のNMDA曝露後に細胞を洗浄することによって次回NMDA刺激時の蛍光強度が減弱されること、および短期間のNMDA曝露後に細胞を洗浄することにより細胞膜上のNMDAR蛋白質の発現量が減少することが明らかとなった。以上の結果はラット大脳皮質由来初代培養神経細胞において、アゴニストが結合することによってではなくアゴニストが解離することによって開始されるNMDARのインターナライゼーションに伴う脱感作が引き起こされる可能性を示唆するものである。NMDARチャネルを介する大量のCa^イオン流入が、多くの神経細胞死出現に深く関与する事実を考慮すると、この脱感作出現メカニズムの解明により、脳内神経細胞死防御を指向する創薬戦略の展開が期待される。研究課題/領域番号:18790051, 研究期間(年度):2006 – 2007出典：「NMDAレセプターチャネルのメンブラントラフィック機構」研究成果報告書　課題番号18790051（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-18790051/)を加工して作

NMDA受容体の脱感作現象に関する分子薬理学的研究

取得学位：博士(薬学)，学位授与番号：博甲第719号，学位授与年月日：平成17年3月22

Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro

AbstractWe have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2’-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells

Activator protein-1 responsive to the group II metabotropic glutamate receptor subtype in association with intracellular calcium in cultured rat cortical neurons

金沢大学大学院自然科学研究科分子作用学Activation of ionotropic glutamate (Glu) receptors, such as N-methyl-d-aspartate receptors, is shown to modulate the gene transcription mediated by the transcription factor activator protein-1 (AP1) composed of Fos and Jun family proteins in the brain, while little attention has been paid to the modulation of AP1 expression by metabotropic Glu receptors (mGluRs). In cultured rat cortical neurons, where constitutive expression was seen with all groups I, II and III mGluR subtypes, a significant and selective increase was seen in the DNA binding activity of AP1 120 min after the brief exposure to the group II mGluR agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) for 5 min. In cultured rat cortical astrocytes, by contrast, a significant increase was induced by a group I mGluR agonist, but not by either a group II or III mGluR agonist. The increase by DCG-IV was significantly prevented by a group II mGluR antagonist as well as by either an intracellular Ca2+ chelator or a voltage-sensitive Ca2+ channel blocker, but not by an intracellular Ca2+ store inhibitor. Moreover, DCG-IV significantly prevented the increase of cAMP formation by forskolin in cultured neurons. Western blot analysis revealed differential expression profiles of Fos family members in neurons briefly exposed to DCG-IV and NMDA. Prior or simultaneous exposure to DCG-IV led to significant protection against neuronal cell death by NMDA. These results suggest that activation of the group II mGluR subtype would modulate the gene expression mediated by AP1 through increased intracellular Ca2+ levels in cultured rat cortical neurons. © 2007

P-Glycoprotein in skin contributes to transdermal absorption of topical corticosteroids

ATP binding cassette transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed in skin, but their involvement in transdermal absorption of clinically used drugs remains unknown. Here, we examined their role in transdermal absorption of corticosteroids. Skin and plasma concentrations of dexamethasone after dermal application were reduced in P-gp and BCRP triple-knockout (Mdr1a/1b/Bcrp−/−) mice. The skin concentration in Mdr1a/1b/Bcrp−/− mice was reduced in the dermis, but not in the epidermis, indicating that functional expression of these transporters in skin is compartmentalized. Involvement of these transporters in dermal transport of dexamethasone was also supported by the observation of a higher epidermal concentration in Mdr1a/1b/Bcrp−/− than wild-type mice during intravenous infusion. Transdermal absorption after dermal application of prednisolone, but not methylprednisolone or ethinyl estradiol, was also lower in Mdr1a/1b/Bcrp−/− than in wild-type mice. Transport studies in epithelial cell lines transfected with P-gp or BCRP showed that dexamethasone and prednisolone are substrates of P-gp, but are minimally transported by BCRP. Thus, our findings suggest that P-gp is involved in transdermal absorption of at least some corticosteroids in vivo. P-gp might be available as a target for inhibition in order to deliver topically applied drugs and cosmetics in a manner that minimizes systemic exposure. © 2017 Elsevier B.V.Embargo Period 12 month

Involvement of the Transporters P-Glycoprotein and Breast Cancer Resistance Protein in Dermal Distribution of the Multikinase Inhibitor Regorafenib and Its Active Metabolites

Regorafenib is a multikinase inhibitor orally administered to colorectal cancer patients, and is known to often exhibit dermal toxicity. The purpose of this study is to clarify possible involvement of P-glycoprotein and breast cancer resistance protein (BCRP) in the dermal accumulation of regorafenib and its active metabolites M-2 and M-5. Following intravenous administration in triple knockout (Abcb1a/1b/bcrp -/-; TKO) and wild-type (WT) mice, delayed plasma clearance of M-2 and M-5, but not regorafenib, was observed in TKO mice compared to WT mice. Elacridar, an inhibitor of both transporters, also caused delayed clearance of M-2 and M-5, suggesting that these transporters are involved in their elimination. Skin-to-plasma concentration ratios of regorafenib, M-2, and M-5 were significantly higher in TKO mice than in WT mice. Elacridar increased skin-to-plasma and epidermis-to-plasma concentration ratios of regorafenib. Basal-to-apical transport of M-2 and M-5 was observed in LLC-PK1-Pgp and MDCKII/BCRP/PDZK1 cells, which was inhibited by elacridar and the BCRP inhibitor Ko143, respectively. The present findings thus indicate that P-glycoprotein and BCRP are involved in the accumulation of regorafenib and its active metabolites in the skin, by affecting either their systemic exposure or their plasma distribution in the circulating blood. © 2017 American Pharmacists Association®.Embargo Period 12 month

Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation

The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [ 3H]ERGO in several brain regions of octn1 -/- mice was much lower than that in wild-type mice, whereas extracellular marker [ 14C]mannitol exhibited similar distribution in the two strains. The [ 3H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [ 3H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [ 3H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development. Crown Copyright © 2012

Utilization of Liver Microsomes to Estimate Hepatic Intrinsic Clearance of Monoamine Oxidase Substrate Drugs in Humans

Purpose: Monoamine oxidases (MAOs) are non-CYP enzymes that contribute to systemic elimination of therapeutic agents, and localized on mitochondrial membranes. The aim of the present study was to validate quantitative estimation of metabolic clearance of MAO substrate drugs using human liver microsomes (HLMs). Methods: Three MAO substrate drugs, sumatriptan, rizatriptan and phenylephrine, as well as four CYP substrates were selected, and their disappearance during incubation with HLMs or mitochondria (HLMt) was measured. Metabolic clearance (CL) was then calculated from the disappearance curve. Results: CL obtained in HLMs for sumatriptan and a typical MAO substrate serotonin was correlated with that obtained in HLMt among ten human individual livers. Hepatic intrinsic clearance (CLint,vitro) estimated from CL in HLMs was 14–20 and 2–5 times lower than in vivo hepatic intrinsic clearance (CLint,vivo) obtained from literature for MAO and CYP substrates, respectively. Utilization of HLMs for quantitatively assessing metabolic clearance of MAO substrates was further validated by proteomics approach which has revealed that numerous proteins localized on inner and outer membranes of mitochondria were detected in both HLMs and HLMt. Conclusion: CLint,vitro values of MAO substrate drugs can be quantitatively estimated with HLMs and could be used for semi-quantitative prediction of CLint,vivo values. © 2017 Springer Science+Business Media New YorkEmbargo Period 12 month