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Effect of Hypoxia, pH, External Ions and Gas Transport on Heart Beat and Contractility in Isolated Single Cardiomyocyte.

Research Project

Project/Area Number 63480114
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 環境生理学(含体力医学・栄養生理学)
Research InstitutionYamagata University

Principal Investigator

DOI Katsuhiko  Yamagata Univ, Dept, of Physiol., Professor, 医学部, 教授 (20002222)

Co-Investigator(Kenkyū-buntansha) KAGAWA Tomoko  Yamagata Univ, Dept, of Physiol., Research fellow, 医学部, 教務職員 (20160971)
MATSUYAMA Kiyoji  Yamagata Univ, Dept, of Physiol., Research fellow, 医学部, 助手 (40209664)
SHIBUYA Izumi  Yamagata Univ, Dept, of Physiol., Research fellow, 医学部, 助手 (50162649)
UCHIDA Katsuo  Yamagata Univ, Dept, of Physiol., Research fellow, 医学部, 助手 (00134114)
Project Period (FY) 1988 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥5,200,000 (Direct Cost: ¥5,200,000)
KeywordsCytosolic pH / Cytosolic Ca / Isolated single cardiomyocyte / Ischemia / Acidosis / Na / H exchanger / Ca exchanger / O_2 diffusion coefficient / Hypoxia / 心筋細胞膜 / ミトコンドリア膜 / O^2拡散速度係数 / 細胞内pH,Ca^<++>濃度同時測定 / Ca^<++>感受性色素Fura2 / Na@H交換輸送系 / Na@Ca交換輸送系 / 単離心筋細胞 / pH感受性色素(BCECF-AM) / アシドーシス / ミオグロビン / CO_2,O_2拡散速度 / 細胞内Ca^<++> / 顕微測光システム
Research Abstract

Method of simultaneous measurement of intracellular pH and Ca_<2+>in an isolated single cardiac myocyte.
We have developed a method of simultaneous measurement of intracellular pH (pH_i) and Ca^<2+> ([Ca^<2+>] _i) in a single myocyte isolated from rat ventrtcle by a microfluorometric technique using intracellular pH and Ca^<2+> indicators (BCECF and Pure-2). We made two optical choppers, one with four-band pass filters to gain four types of exctaion wavelength (BCECF : 500, 400 nm, Fura-2 : 340, 380 nm), and the other had two filter cassettes to utilize two types of fluorescence (BCECF : > 520 nm, Fura-2 : > 420 nm). Alternate measurements of pH_i and (Ca^<2+>) _i were possible with computer control of the motion of two choppers. After loading two dyes into freshly isolated myocytes, pH and Ca of the intracellular dyes were calibrated using a nigericin calibration technique for pH and an ionomycin calibration technique for Ca. The ratios of intensities emitted at two excitation waveleng … More ths (BCECF : 500/440 nm, Fura-2 : 340/380 nm) were very sensitive to pH change between pH 6.5 and 8.0, and Ca change between pCa 5.0 and 7.0. It was found that pH_i and (Ca^<2+>) _i can be calculated according to equations obtained empirically in this experiment : pH= 7.15 + log ((ratio-0.7)/(5.6-ratio)), and pCa =5.7 + log ((4.3-ratio)/(ratio-0.43)}. In our system, it was found that the fluorescence signal of BCECF was not influenced by Ca concentration from Ca-free to l mM, and the Fura-2 signal was not influenced with pH greater then 6.5.
Interconnection of intracellular pH and Ca^<2+> changes in a single myocyte from rat ventricle.
To elucidate the influence of intracellular acidosis on the contraction mechacism of cardiac muscle, we made simultaneous measurements of intracellular pH (pH_i) and Ca^<2+> ((Ca^<2+>) _i) in a single myocyte using a microflurometric method. We used a NH_4Cl prepulse technique to achieve intracellular acidosis. After removal of NH_4Cl solution, pH_i decrease and sequential pH_i recovery rate from minimum pH_i (the mean of the activity of Na^+/H^+ exchanger) were enhanced in a NH_4Cl dose-dependent manner. In a simultaneous measurement, (Ca^<2+>) _i increases corresponded to the pH_i recovery phase, and the degree of (Ca^<2+>) _i increase was enhanced according to the degree of acidosis. Removal of NH_4Cl with Ca-free solution had no effect on (Ca^<2+>) increase. By addition of amiloride (300 muM), both pHi recovery and [Ca^<2+>] _i, increase were blocked. When NH_4Cl was washed out with solution containing ouabain (70 muM), further increase of [Ca^<2+> _i occurred. These results suggested that these [Ca^<2+>] _i increases were due to Ca^<2+> influx through Na^+/Ca^+ exchangers coupled with Na^+/H^+ exchangers activated by intracellular acidosis, and that this mechanism might enhance the contraction mechanism of cardiac muscle.
Estimation of the oxygen diffusion coefficient (Do_2)
The Do2 at 37 <@D10@>D1 was 2.9 (] SY.+-.[) 0.05 x 10<@D1-6@>D1 cm<@D12@>D1/s for plasma membrane and 2.2 (] SY.+-.[) 0.10 x 10<@D1-6@>D1 cm<@D12@>D1/s for mitochondrial membrane of rat cardiomyocytes (p < 0.001). Oxygen defusion was measured as changes in O<@D22@>D2-induced fluorescence quenching of a hydrophobic probe, pyrenebutyric acid. These are the first reported measurements of Do<@D22@>D2 in cardiomyocyte membranes. Moreover, the Do<@D22@>D2 value for plasma membrane significantly reduced to 2.5 (] SY.+-.[) 0.08 x 10<@D1-6@>D1 cm<@D12@>D1/s by 15 min reperfusion after 30 min ischemia. A specific structural change in the membranes seems to be responsible for the observed Do<@D22@>D2 reduction on reperfusion. Less

Report

(4 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • 1988 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] 土居 勝彦: "単一心筋細胞の収縮力に及ぼす低酸素、pH、Ca^<++>の影響とガス交換機能" 平成元年度 受託研究報告書(山形大学医学部). 1-20 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Matsuyama,K.,Shibuya,I.Tanaka,K.and Doi,K.: "Influence of extracellular acidosis and temperature on intracellular pH and Ca^<2+> in a single myocyte isolated from rat ventricle。" Int.J.Biometeorol.34. 130-130 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Uchida,K.Kagawa,T.and Doi,K.: "Effect of temperature on the oxygen transporting ability of myoglobin." Int.J.Biometeorol.34. 130-130 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Shibuya,I.,Matsuyama,K.Tanaka,K。and Doi,K.: "A microfluorometric method for simultaneous measurement of changes in cytosolic free calcium concentration and pH in single cardiac myocytes." Jpn.J.Physiol.(1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Uchida,K.,Matsuyama,K.,Tanaka,K.,and Doi,K.: "Diffusion coefficient of oxygen in plasma and mitochondrial membrance of rat cardiomyocytes." Jpn.J.Physiol.(1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Uchida, K., Matsuyama, K., Matsuyama, K., Niizeki, K., Shibuya, I., and Doi, K.: "Gad kinetics in single isolated myocytes - role of myoglobin." Int. J. Biometerol.33. 203-204, (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Matsuyama, K., Uchida, K., Niizeki, K., Shibuya, I., and Doi, K. :"Intracellular pH change in a single myocyte isolated from guinea pig ventricle." Int. J. Biometeorol.33. 204, (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Matsuyama, K., Shibuya, I., Tanaka, K., and Doi, K. :"Influence of extracellular acidosis and temperature on intracellular pH and Ca^<2+> in a single myocyte isolated from rat ventricle." Int. J. Biometeorol.34. 130, (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Uchida, K., Kagawa, T., and Doi, K.: "Effect of temperature on the oxygen transporting ability of myoglobin." Int. J. Biometeorol.34. 130, (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Shibuya, I., Matsuyama, K., Tanaka, K, and Doi, K. :"A microfluorometric method for simultaneous measurement of changes in cytosolic free calcum concentration and pH in single cardiac myocytes." Jpn. J. Physiol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Uchida, K., Matsuyama, K., Tanaka, K., and Doi, K. :"Diffusion coefficient of oxygen in plasma and mitochondrial membranes of rat cardiomyocytes." Jpn. J. Phydiol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 土居 勝彦: "単一心筋細胞の収縮力に及ぼす低酸素、pH、Ca^<++>の影響とガス交換能" 平成元年度受託研究報告書(山形大学医学部). 1-20 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Matsuyama.K.,Shibuya,I.Tanaka,K.and Doi,K.: "Influence of extracellular acidosis and temperature on intracellular pH and Ca^<2+> in a single myocyte isolated from rat ventricle。" Int.J.Biometeorol.34. 130-130 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Uchida,K.,Kagawa,T.and Doi,K.: "Effect of temperature on the oxygen transporting ability of myoglbin." Int.J.Biometeorol.34. 130-130 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Shibuya,I.,Matsuyama,K.Tanaka,K.and Doi,K.: "A microfluorometric method for simultaneous measurement of changes in cytosolic free calcium concentration and pH in single cardiac myocytes." Jpn.J.Physiol.(1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Uchida,K.,Matsuyama,K.,Tanaka,K.,and Doi,K.: "Diffusion coefficient of oxygen in plasma and mitochondrial membranes of rat cardiomyocytes." Jpn.J.Physiol.(1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 土居勝彦,渋谷泉,松山清治,内田勝雄,加川朋子: "アシド-シスと心機能" 病態生理. 9. 869-877 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Uchida,K.,Matsuyama,K.,Niizeki,K.,Shibuya,I.,and Doi,K.: "Gas Kinetics in sigle isolated myocytes-role of myoglobin." Int.J.Biometeorol. 33. 203-204 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Matsuyama,K.,Uchida,K.,Niizeki,K.,Shibuya,I.,and Doi,K.: "Intracellular pH change in a single myocyte insolated from guinea pig ventricle." Int.J.Biometeorol. 33. 204-204 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Doi,K.;Shibuya,I.;Matsuyama,k.: Japan Journal Physiology. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Doi,K.;Shibuya,I.;Matsuyama,k.: Japan Journal Physiology. (1989)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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