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

本研究应用膜片钳技术和荧光染料，测定了离体细胞的离子电流，并探讨了细胞间钙离子的动力学性质。倒置显微镜（Nikon，TMD）配备有氙灯和用于紫外光的滤光器，并放置在无振动台（Meiritsu Co.，AD-107）。在本项目的第一次制备中，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。从鸡中酶促分离c细胞，并应用贴片电极。当刺激去极化时，细胞可产生动作电位。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诱发的长丢失内向电流来阐明 ...更多信息 在等渗Ba^2+盐水中。这些c细胞被抗钙离子抗体（#770lRl）染色呈阳性。一些细胞不产生动作电位，抗体染色微弱或可忽略不计。作为第二种制备方法，我们研究了一个已建立的正常人胚胎肝细胞系（HuL-1）。当ACh 10 μ M局部作用于该细胞时，膜电位出现突然的超极化。在-40 mV电压钳下，ACh诱发外向电流。电流是由细胞内Ca^&lt;2+&gt;介导的，因为使用含Ca^&lt;2+&gt;小于10 μ M的内部盐水<-8>会减弱电流，而应用Ca^&lt;2+&gt;-离子载体（A23187，10 μ M）则诱发类似的外向电流。这一结论用Ca^2+敏感的荧光染料Fura-2得到了证实。然后施加ACh，一致地观察到来自Fura-2负载的细胞的荧光强度（在340 nm处激发）的增加。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