| Project/Area Number |
09670087
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | Akita University |
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Principal Investigator |
IIJIMA Toshihiko Akita Univ.Sch.Med, Dept.Pharmacol, Professor, 医学部, 教授 (30004724)
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| Co-Investigator(Kenkyū-buntansha) |
ONO Kyoichi Akita Univ.Sch.Med, Dept.Pharmacol, Assoc.Professor, 医学部, 助教授 (70185635)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Vascular Endothel / Intracellular Ca^<2+> concentrarion / Fura-2 / Capacitative Ca^<2+> Entry Chanell / Patch Clamp / Cl^- Current / Fluid-stream / trp / Cl^-電流 / trp / Fura‐2 |
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| Research Abstract |
In cultured human aortic endothelial cells (HAECs) histamine (1-100 muM) produced a biphasic response in [Ca^<2+>]_i. We conducted whole-cell current recording combined with fluorescence measurement of intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in order to investigate the mechanism underlying the C1-sensitive Ca^<2+> entry in endothelial cells. Membrane currents from single endothelial cells were measured using nystatin-perforated patch clamp technique. The histamine-induced Ca^<2+> entry is inhibited reversibly either by decreasing the extracellular concentration of Cl^- or by Cl^- channel blockers. Histamine activated an outward current, followed by a sustained inward current at -50 mV.The reversal potential was more negative than -60 mV for the initial outward current and approximately -30 mV for the sustained inward current with the normal Tyrode solution and the internal solution containing 30 epsilonlm Cl^-. The fluid stream applied through a micro tube also induced an inc
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rease in [Ca^<2+>]_i, which was dependent on both the flow rate and the extracellular Ca^<2+> concentration. The fluid stream-induced increase in [Ca^<2+>]_i was accompanied by the activation of an inward current at -53 mV.The reversal potential of the fluid stream-induced current shifted to positive potentials by reducing the external Cl^- concentration. Results from the effects of histamine and a fluid-stream we concluded that not only the histamine-induced inward current but also the fluid stream-induced current is carried mainly by Cl^-. And Cl^- current plays a crucial role in modulating the Ca^<2+>_i influx by altering the membrane potential of endothelial cells.
It is well known that sulfonylureas, which block ATP-sensitive K^<2+> channels (KATP), inhibit cystic fibrosis transmembrane regulator (CFTR) Cl^- channels, volume-sensitive Cl^- channels and Ca^<2+> activated Cl^- channels in epithelial and cardiac cells. Glibenclamide (10-500 muM) has little effect on the initial transient increase in [Ca^<2+>]_i induced by histamine but inhibited the following sustained increase in [Ca^<2+>]_i in a concentration-dependent manner. The IC50 value was 151.8 muM and the Hill's coefficient was 1.9. The sustained increase in [Ca^<2+>]_i induced by ATP (100 muM) and cyclopiazonic acid (10 muM) were also suppressed by glibenclamide, Under the patch-clampcondition, however, glibenclamide suppressed the histamine-induced Cl^- current with an IC50 value of 12 muM.Histamine-induced Cl^- current was almost completely abolished by 100 muM glibenclaminde but the sustainedincrease in [Ca^<2+>]_i was only partially suppressed. Thus, the glibenclamide-induced inhibition of the Ca^<2+> entry was not due to possible changes of membrane potential by suppression of Cl^- current, but due to direct effects on the Ca^<2+> influx pathway. The present results indicate that glibenclamide disturbs the Ca^<2+> influx pathway independent of the inhibition of Cl^- current, However, the activation of Cl^- channels might be functionally coupled with Ca^<2+> influx pathway in vascular endothelial cells. Less
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