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

利用膜片钳技术和荧光染料研究细胞内钙离子的动态特性。

基本信息

批准号：
62870002
负责人：
KAWA Kazuyoshi
金额：
$ 4.54万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

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

在这项研究中，我们使用膜片钳技术和荧光染料，试图测量一些分离的细胞内的离子电流，并研究细胞内钙离子的动态特性。倒置显微镜(尼康，TMD)配备了氙气灯和紫外线滤光片，放置在无震动的桌子上(美力通公司，AD-107)。在这个项目的第一个准备中，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钙离子浓度升高而增加激素分泌的特征。用酶法分离鸡的c-细胞，并应用贴片电极。当被刺激去极化时，细胞可以产生动作电位。动作电位主要由Na~+-通道和延迟整流性K~+-通道的激活引起。通过观察引起…的长时间丢失的内向电流，也阐明了钙离子通道的参与更多的是等渗的生理盐水。这些c细胞与抗降钙素抗体(#7701Rl)呈阳性反应。一些细胞不产生动作电位，抗体染色模糊或可忽略不计。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，我们研究了一个已建立的人胚胎(HUL-1)肝细胞系，当ACh 10um局部作用于该细胞时，该细胞的膜电位呈现突然的超极化。在-40 mV的电压钳下，施加ACh产生外向电流。该电流是由细胞内钙离子介导的，因为使用含钙小于10^&lt；-8&gt；M的内盐水可使电流减弱，而使用钙离子载体(A23187，10 um)可引起类似的外向电流。这一结论得到了钙离子敏感的荧光标记Fura-2的证实。加入ACh后，Fura-2负载细胞的荧光强度(激发波长为340 nm)持续增强。较少