カルシウムイメージングとパッチクランプによる緑内障治療薬の神経保護作用の検証

Material type: 文書・図像類

Author: 佐々木, 次壽ほか

Publisher: 金沢大学附属病院

Publication date: 2006-03

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Note (General)：: 1)パッチクランプ法による神経節細胞Ca電流への緑内障治療薬の効果:金魚網膜を酵素的に処理し神経節細胞を単離,顕微鏡下に同定した.穿孔パッチクランプをかけwhole cell configurationにてCa電流を測定した.網膜神経節細胞L-型Caチャネルへのニルバジピンの最小有効濃度が1μMであ...

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Material Type: 文書・図像類
Title: カルシウムイメージングとパッチクランプによる緑内障治療薬の神経保護作用の検証
Author/Editor: 佐々木, 次壽
Sasaki, Tsugihisa
Author Heading: 佐々木, 次壽
Sasaki, Tsugihisa
Publication, Distribution, etc.: 金沢大学附属病院
Publication Date: 2006-03
Publication Date (W3CDTF): 2006-03
Alternative Title: The research in neuroprotection effect of anti-glaucoma drug with Ca imaging and patch clamping
Periodical title: 平成17(2005)年度科学研究費補助金基盤研究(C) 研究成果報告書 = 2005 Fiscal Year Final Research Report
No. or year of volume/issue: 2004-2005
Volume: 2004-2005
Pages: 3p.-
Text Language Code: jpn
Subject Heading: ニルバジピン
パッチクランプ
神経節細胞
網膜
電位依存性Caチャネル
神経節細胞選択的Caイメー
NMDA受容体
低酸素応答
Nilvadipine
Patch clamp
Ganglion cell
Retina
Voltage-gated Ca channel
Ganglion cell selective Ca imaging
NMDA receptor
Hypoxia induced response
Target Audience: 一般
Note (General): 1)パッチクランプ法による神経節細胞Ca電流への緑内障治療薬の効果:金魚網膜を酵素的に処理し神経節細胞を単離,顕微鏡下に同定した.穿孔パッチクランプをかけwhole cell configurationにてCa電流を測定した.網膜神経節細胞L-型Caチャネルへのニルバジピンの最小有効濃度が1μMであること,保持電位が-28〜+50mVでCa電流の抑制効果を認めることを認めた.これらの結果と内服投与後の眼内濃度よりニルバジピンは神経節細胞に直接作用して保護しうると推測された.これらの結果はJournal of ocular pharmacology and therapeuticsに平成17年12月に投稿した.2)Caイメージング法による神経節細胞の低酸素負荷による細胞内Ca^<2+>濃度([Ca^<2+>]_1)上昇の解析.ラットを用いて,新たに開発した網膜スライス内神経節細胞(GC)選択的Caメージング法を用いて神経節細胞内Ca濃度測定を行った.スライス標本を低O_2条件にするとGCの[Ca^<2+>]_1上昇が認められた,GC(0.14±0.04, Mean±SD)。この[Ca^<2+>]_i上昇はNMDA型glu受容体阻害薬APV(60μM)でGCにおいて樹上突起部で抑制された,神経節細胞層(0.12±0.01,), IPL(0.04±0.04, p<0.005).CNQXはGCの[Ca^<2+>]_i上昇を抑制しなかった.L型Caチャネル阻害薬ニフェジピンはGCでは抑制しなかった(0.13±0.01).結論:低酸素条件下でのGC樹上突起部での[Ca^<2+>]_I)上昇はNMDA受容体を介する.これらの結果はJapanese Journal of Ophthalmologyに平成18年3月に投稿予定である.
1) The effect of nilvadipine on the voltage-gated Ca channel of retinal ganglion cells (GC).GC were enzymatically dissociated from isolated retina. Cells were recorded by perforated-patch clamp technique in whole cell configuration The current-voltage relation for the nilvadipine-sensitive current was bell-shaped and the peak current reached a maximum at -8 mV in the presence and absence of nilvadipine. Nilvadipine block of voltage-gated Ca current was dose-dependent between 1 and 100 μM. The inhibitory actions of nilvadipine on Ca channels could relieve the intracellular Ca increase in GCs and rescue the GCs from death in glaucoma.2) Elevation of intracellular Ca^<2+> concentration ([Ca^<2+>]_i) induced by hypoxia in ganglion cellsApplication of fluo-3 to the cut edge of the optic nerve of 6-week-old rats. The Ca images of sliced retina were captured. A hypoxic condition was created by superfusing the retinal slice with a solution with an oxygen/glucose deprived solution.Results : The retrograde staining method stained GC selectively. Fifteen minutes of hypoxic conditions induced [Ca^<2+>]_i increases in GC (Δ0.13±0.03, n=23). Application of 60 μMDL-2-amino-5-phosphonovaleric acid counteracted the hypoxia-induced [Ca^<2+>]_i increase in dendrites partially (Δ0.03±0.02, n=4, p<0.005), but not in somata (Δ0.12±0.02, n=9). GC dendrites showed a further increase in [Ca^<2+>]_i after switching back to oxygenated solution (Δ0.14±0.04, n=4). Neither 6-cyano-7-nitroquinoxaline-2,3-dione disodium, DL-threo-β-benzyloxyaspartate, nifedipine nor bepridil inhibited the hypoxia-induced [Ca^<2+>]_i increase. Ca^<2+>-free superfusion prevented the anoxia-induced [Ca^<2+>]_i increase in somata (Δ0.07±0.02, n=5, p<0.005), not in dendrites (Δ0.16±0.005, n=4).Conclusions : The mechanisms of hypoxia-induced increase in [Ca^<2+>]_i differ between somata and dendrites. The NMDA channel of dendrites seems to be the main route of Ca^<2+> influx.
研究課題/領域番号:16591741, 研究期間(年度):2004-2005
出典：「カルシウムイメージングとパッチクランプによる緑内障治療薬の神経保護作用の検証」研究成果報告書　課題番号16591741 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作成
DOI: 10.24517/00056946
Access Restrictions: 限定公開
Related Material: https://kaken.nii.ac.jp/search/?qm=00338188
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16591741/
https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-16591741/165917412005kenkyu_seika_hokoku_gaiyo/
Data Provider (Database): 国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）
https://irdb.nii.ac.jp
Original Data Provider (Database): 金沢大学 : 金沢大学学術情報リポジトリKURA