1999 Fiscal Year Annual Research Report

血管の細胞シグナル伝達網制御を多項目連続測定可能な光学システムの開発

Research Project

Project/Area Number	10557072
Research Institution	Kyushu University
Principal Investigator	金出英夫九州大学, 大学院・医学系研究科, 教授 (80038851)
Co-Investigator(Kenkyū-buntansha)	平野勝也九州大学, 大学院・医学系研究科, 講師 (80291516) 西村淳二九州大学, 大学院・医学系研究科, 助教授 (90237727)
Keywords	細胞シグナル伝達 / 血管内皮細胞 / 血管平滑筋細胞 / fura-2 / BCECF / 光学線維束 / 血管緊張調節
Research Abstract	Ca^<2+>指示蛍光色素(Fura-2)とpH指示蛍光色素(BCECF)を、ラット大動脈初代培養平滑筋細胞に負荷し、細胞質Ca^<2+>濃度([Ca^<2+>]i)とpH変化の同時連続測定システムの開発を試みた。表面蛍光測定には我々が開発した装置(CAM230-OF3)を用いた。Fura-2蛍光(500nm)比(Ratio=F340/380)とBCECF蛍光(540nm)比(Ratio=F500/450)の記録には、光学線維束を用いた。 1.Fura-2とBCECFを二重負荷した細胞におけるに[Ca^<2+>]i測定二重負荷した細胞の[Ca^<2+>]iを測定したところ、Fura-2蛍光(500nm)比(Ratio=F340/380)の変化は Fura-2単独負荷の場合と同様であった。逆に、BCECF単独を負荷した細胞で、[Ca^<2+>]iを変化させても、Fura-2蛍光(500nm)比(Ratio=F340/380)値の変化は認められなかった。 2.Fura-2とBCECFを二重負荷した細胞におけるpH測定二重負荷した細胞のpHを変え、BCECF蛍光(540nm)比(Ratio=F500/450)を測定した。蛍光比の変化はBCECF単独負荷の場合と全く同様であった。逆に、Fura-2単独負荷細胞で、pHを変化させても蛍光比の変化は認められなかった。 3.すなわち、Fura-2とBCECFを二重負荷した細胞における[Ca^<2+>]i測定とpH測定では、色素同志の干渉は無い事が判明した。 4.以上の結果から、2台のCAM230をそれぞれに[Ca^<2+>]i測定とpH測定条件に設定し、高速測定切替えを行えば[Ca^<2+>]iと細胞質pH変化の同時測定が可能であるという実験的根拠を得ることができた。高速に2つの測定系を切り替えるには、光学線維束の試料側端あるいは光源側端を交互に遮蔽する装置を開発することで達成可能である。今後はこの方向で開発を続ける。

Research Products

(11 results)

All Other

All Publications (11 results)

[Publications] Hirano M: "Expression,subcellular localization and cloning of the 130 kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells."Biochem Biophys Res Commun. 254. 490-496 (1999)
[Publications] Yasutsune T: "Vasorelaxation and inhibition of the voltage-operated Ca^<2+> channels by FK506 in porcine coronary artery."Br J Pharmacol. 126. 717-727 (1999)
[Publications] Zhou YB: "The exogenously added small subunit of smooth muscle myosin phosphatase increases the Ca^<2+> sensitivity of the contractile apparatus in the permeabilized porcine renal artery."Biochem Biophys Res Commun. 254. 158-163 (1999)
[Publications] Zhou YB: "NH_2-terminal fragments of 130 kDa subunit of myosin phoshatase increases Ca^<2+> sensitivity of the porcine renal artery."J Physiol. 516. 55-65 (1999)
[Publications] Kawasaki J: "Troglitazone inhibits the capacitative Ca^<2+> entry in endothelial cells."Eur J Pharmacol. 373. 111-120 (1999)
[Publications] Ihara E: "Thapsigargin-induced endothelium dependent triphasic regulation of vascular tone in the porcine renal artery.:-,1999"Br J Pharmacol. 128. 689-699 (1999)
[Publications] Ushio-Fukai M: "The mechanism for the decrease in cytosolic Ca^<2+> concentrations induced by angiotensin II in the high K^+-depolarized rabbit femoral artery."Br J Pharmacol. (in press).
[Publications] Ushio-Fukai M: "Changes in the cytosolic Ca^+ concentration and Ca^<2+>-sensitivity of the contractile appatatus during angiotensin II-induced desensitization in the rabbit femoral artery."Br J Pharmacol. (in press).
[Publications] Mizuno O: "Regulation of thrombin receptor activity in endothelial cells in situ"Eur J Pharmacol. (in press).
[Publications] Ihara E: "The mechanism of bradykinin-induced endothelium-dependent contraction and relaxation in the porcine interlobar renal artery."Br J Pharmacol. (in press).
[Publications] Kanaide H: "Method in Molecular Biology,Vol.114:Calcium Signaling Protocols,Edited by:DG Lambert"Humana Press Inc.,Totowa,NJ. 9 (1999)

1999 Fiscal Year Annual Research Report

血管の細胞シグナル伝達網制御を多項目連続測定可能な光学システムの開発

Principal Investigator

金出 英夫 九州大学, 大学院・医学系研究科, 教授 (80038851)

Research Products

[Publications] Hirano M: "Expression,subcellular localization and cloning of the 130 kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells."Biochem Biophys Res Commun. 254. 490-496 (1999)

[Publications] Yasutsune T: "Vasorelaxation and inhibition of the voltage-operated Ca^<2+> channels by FK506 in porcine coronary artery."Br J Pharmacol. 126. 717-727 (1999)

[Publications] Zhou YB: "The exogenously added small subunit of smooth muscle myosin phosphatase increases the Ca^<2+> sensitivity of the contractile apparatus in the permeabilized porcine renal artery."Biochem Biophys Res Commun. 254. 158-163 (1999)

[Publications] Zhou YB: "NH_2-terminal fragments of 130 kDa subunit of myosin phoshatase increases Ca^<2+> sensitivity of the porcine renal artery."J Physiol. 516. 55-65 (1999)

[Publications] Kawasaki J: "Troglitazone inhibits the capacitative Ca^<2+> entry in endothelial cells."Eur J Pharmacol. 373. 111-120 (1999)

[Publications] Ihara E: "Thapsigargin-induced endothelium dependent triphasic regulation of vascular tone in the porcine renal artery.:-,1999"Br J Pharmacol. 128. 689-699 (1999)

[Publications] Ushio-Fukai M: "The mechanism for the decrease in cytosolic Ca^<2+> concentrations induced by angiotensin II in the high K^+-depolarized rabbit femoral artery."Br J Pharmacol. (in press).

[Publications] Ushio-Fukai M: "Changes in the cytosolic Ca^+ concentration and Ca^<2+>-sensitivity of the contractile appatatus during angiotensin II-induced desensitization in the rabbit femoral artery."Br J Pharmacol. (in press).

[Publications] Mizuno O: "Regulation of thrombin receptor activity in endothelial cells in situ"Eur J Pharmacol. (in press).

[Publications] Ihara E: "The mechanism of bradykinin-induced endothelium-dependent contraction and relaxation in the porcine interlobar renal artery."Br J Pharmacol. (in press).

[Publications] Kanaide H: "Method in Molecular Biology,Vol.114:Calcium Signaling Protocols,Edited by:DG Lambert"Humana Press Inc.,Totowa,NJ. 9 (1999)

金出英夫九州大学, 大学院・医学系研究科, 教授 (80038851)