|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1993 : ¥1,300,000 (Direct Cost : ¥1,300,000)
To measure intracellular Ca^<2+> concentration ( [Ca^<2+>] _i) in skeletal muscle fibers at rest, the Ca^<2+> indicator, fura-2, conjugated to dextran (fura dextran, MW -10,000) was injected into single twitch fibers of frogs, and the indicator's Ca^<2+> -dependent fluorescence in the cytoplasm was measured at 17ﾟC.
Calibration of the indicator fluorescence (in terms of [Ca^<2+>] _i) was carried out in the muscle fibers treated with beta-escin to permeabilize the cell membrane. After the treatment with 5 muM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca^<2+>, ATP) , while the 10 kDa fura dexran only slowly leaked out of the cell. The major fraction of cytoplasmic proteins (14-80 kDa) was retained in the beta-escin-treated cells, as only a trace amount of proteins was detected in the exracellular solution samples by SDS-PAGE with silver staining. It was thus possible to estimate the calibration parameters of the indicator fluorescence in the cell (in the presence of cellular proteins) by changing the Ca^<2+> concentration in the bething solution to various levels. When [Ca^<2+>] of the bathing solution was changed to pCa>9 to higher levels (pCa7-4), the Ca^<2+> -dependent fluorescence of fura dextran changed to a new steady level within a few minutes. The indicator's dissociation constant for Ca^<2+> (K_D) estimated in the cell was 1.0 muM,which is two-fold higher than that obtained in vitro (0.52 muM). The second method to estimate the K_D, the kinetic analysis of the indicator fluorescence change following electrical stimulation in intact muscle fibers, gave an estimated value of 2.1 muM.The K_D values thus estimated in the cell interior (1.0-2.0 muM) is two- to four-fold higher than that obtained in vitro (0.52 muM). From the parameters estimated in the fibers, we conclude that the resting [Ca^<2+>]_i in frog skeletal muscle fibers is likely in the range of 55-155 nM.