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

在这项研究中，我们使用膜片钳技术和荧光染料，试图测量一些分离细胞中的离子电流，并研究细胞间钙离子的动态特性。倒置显微镜（Nikon， TMD）配有氙灯和紫外线滤光片，放置在无振动台上（meiitsu Co., AD-107）。在本项目的第一个制备中，我们使用了鸡的降钙素分泌细胞（c细胞），因为这些细胞具有响应血清Ca^<2+>浓度升高而增加激素分泌的特征。用酶法从鸡体内分离c细胞，并应用贴片电极。细胞在去极化刺激下可产生动作电位。动作电位主要由Na^+通道和延迟整流K^+通道激活引起。Ca^<2+>-通道的参与也通过观察Ba^<2+>等渗盐水中引起的长期丢失的内向电流来澄清。这些c细胞被抗降钙素抗体（#770lRl）阳性染色。一些细胞不产生动作电位，抗体染色微弱或可忽略。这些细胞可能代表腺体中的非分泌细胞或支持细胞。作为第二种制备，我们研究了从正常人胚胎（hl -1）中获得的已建立的肝细胞细胞系，当ACh 10 uM局部作用于细胞时，膜电位表现出突然的超极化。在- 40mv电压箝位下，施加ACh感应向外电流。电流是由细胞内Ca^<2+>介导的，因为使用含有Ca^<2+>小于10^<-8> M的生理盐水会减弱电流，而使用Ca^<2+>-离子离子（A23187, 10 uM）会引起类似的外电流。这一结论得到了Ca^<2+>-敏感荧光剂Fura-2的证实。然后施加ACh，可以观察到负载Fura-2细胞的荧光强度持续增加（在340 nm处激发）。少

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