| 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)
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| 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
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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
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