Studies on dynamic properties of intracellular calcium ions using patch-clamp technique and fluorescent dyes.

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In this study using patch-clamp techniques and fluorescent dyes, we have tried to measure ionic currents in a number of isolated cells and to investigete dynamic properties of interacellular calcium ions. An inverted microscope (Nikon, TMD) was equipped with a xenon lamp and optical filters for ultraviolete lights and was settled on a vibration-free table (Meiritsu Co., AD-107).For the first preparation in this project, calcitonin-secreting cells (c-cells) of the chick were employed, because these cells have characteristic features of increasing hormonal secretion in response to elevated serum Ca^<2+> concentration. The c-cells were enzymatically isolated from the chick and patch-electrodes were applied. The cells could generate action potentials when stimulated for depolarization. Action potentials were mainly caused by activation of Na^+-channels and delayed rectifier K^+-channels. The involvement of Ca^<2+>-channels was also clarified by observing long-losting inward currents evoked … More in isotonic Ba^<2+> saline. These c-cells were positively stained with anti-calciton antibodies (#770lRl). Some cells generated no action potentials and were stained faint or negligible with the antibodies. These cells may represent non- secreting cells or supporting cells in the gland.As the second preparation, an established cell line of hepatocytes, which has been derived from a normal human embryo (HuL-1), was studied, When ACh 10 uM was locally applied to the cell, the membrane potential showed an abrupt hyperpolarization. Under voltage-clamp at -40 mV, application of ACh induced outward currents. The currents were mediated by intracellular Ca^<2+>, since use of internal saline containing Ca^<2+> less than 10^<-8> M diminished the currents, while application Ca^<2+>-ionophore (A23187, 10 uM) evoked similar outwardcurrents. This conclusion was confirmed using Ca^<2+>-sensitive fluorescent bye, Fura-2. Then ACh was applied, an increase of the intensity of fluorescence (excitation at 340 nm) form Fura-2 loaded cells was consistently observed. Less