Takeuchi Keita, Yoshii Kiyonori
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Hibikino 2-4, Kitakyushu 808-0196, Japan.
Chem Senses. 2008 Jun;33(5):425-32. doi: 10.1093/chemse/bjn009. Epub 2008 Mar 3.
Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.
荧光黄CH（LY）是一种不能透过细胞膜的荧光细胞标记染料，在电生理研究后，人们通常通过记录电极将其导入细胞，以便在显微镜下观察这些细胞，而未考虑其药理作用。最近，我们发现，为了进行显微镜观察而对加载了LY的细胞进行光照会产生不明自由基，这些自由基会不可逆地延迟电压门控Na⁺电流的失活（Higure Y等人，2003年）。在此，我们表明，超氧化物歧化酶（一种分解超氧阴离子的酶）可逆转这种延迟效应，这证实了超氧阴离子就是这种不明自由基。在记录电极中（无细胞质），超氧阴离子的估计平均寿命约为6分钟，因此，即使在黑暗中，当LY在加载到细胞之前受到光照时，Na⁺电流也会延迟。超氧阴离子对电压门控Cl⁻电流没有影响。这些结果表明，超氧阴离子对离子通道的作用具有相当的选择性。本文还讨论了细胞内超氧阴离子的分解以及内源性超氧阴离子对超氧阴离子敏感通道的影响。
