Ikeuchi Yukiko, Kogiso Haruka, Hosogi Shigekuni, Tanaka Saori, Shimamoto Chikao, Inui Toshio, Nakahari Takashi, Marunaka Yoshinori
Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Laboratory of Pharmacotherapy, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan.
J Physiol Sci. 2018 Mar;68(2):191-199. doi: 10.1007/s12576-018-0591-y. Epub 2018 Jan 13.
MQAE is a 'non-ratiometric' chloride ion (Cl)-quenched fluorescent indicator that is used to determine intracellular Cl concentration ([Cl]). MQAE-based two-photon microscopy is reported to be a useful method to measure [Cl], but it is still controversial because a change in cell volume may alter the MQAE concentration, leading to a change in the fluorescence intensity without any change in [Cl]. In an attempt to elucidate the effect or lack of effect of cell volume on MQAE concentration, we studied the effects of changes in cell volume, achieved by applying different levels of osmotic stress, on the intensity of MQAE fluorescence in airway ciliary cells. To study solely the effect of changes in cell volume on MQAE fluorescence intensity, i.e., excluding the effect of any change in [Cl], we first conducted the experiments in a Cl-free nitrate (NO) solution to substitute NO (non-quenching anion for MQAE fluorescence) for Cl in the intracellular fluid. Hypo- (- 30 mM NaNO) or hyper-osmotic stress (+ 30 mM NaNO) effected changes in cell volume, but the stress did not result in any significant change in MQAE fluorescence intensity. The experiments were also carried out in Cl-containing solution. Hypo-osmotic stress (- 30 mM NaCl) increased both MQAE fluorescence intensity and cell volume, while hyper-osmotic stress (+ 30 mM NaCl) decreased both of these properties. These results suggest that the osmotic stress-induced change in MQAE fluorescence intensity was caused by the change in [Cl] and not by the MQAE concentration. Moreover, the intracellular distribution of MQAEs was heterogeneous and not affected by the changes in osmotic stress-induced cell volume, suggesting that MQAEs are bound to un-identified subcellular structures. These bound MQAEs appear to have enabled the measurement of [Cl] in airway ciliary cells, even under conditions of cell volume change.
MQAE是一种“非比率型”氯离子（Cl）淬灭荧光指示剂，用于测定细胞内Cl浓度（[Cl]）。据报道，基于MQAE的双光子显微镜是测量[Cl]的一种有用方法，但仍存在争议，因为细胞体积的变化可能会改变MQAE浓度，导致荧光强度发生变化，而[Cl]却没有任何变化。为了阐明细胞体积对MQAE浓度的影响或无影响，我们研究了通过施加不同程度的渗透应激实现的细胞体积变化对气道纤毛细胞中MQAE荧光强度的影响。为了仅研究细胞体积变化对MQAE荧光强度的影响，即排除[Cl]任何变化的影响，我们首先在无Cl的硝酸盐（NO）溶液中进行实验，用NO（对MQAE荧光无淬灭作用的阴离子）替代细胞内液中的Cl。低渗（-30 mM NaNO）或高渗应激（+30 mM NaNO）引起细胞体积变化，但应激并未导致MQAE荧光强度发生任何显著变化。实验也在含Cl的溶液中进行。低渗应激（-30 mM NaCl）增加了MQAE荧光强度和细胞体积，而高渗应激（+30 mM NaCl）则降低了这两个特性。这些结果表明，渗透应激诱导的MQAE荧光强度变化是由[Cl]的变化引起的，而不是由MQAE浓度引起的。此外，MQAE的细胞内分布是异质的，不受渗透应激诱导的细胞体积变化的影响，这表明MQAE与未鉴定的亚细胞结构结合。这些结合的MQAE似乎使得即使在细胞体积变化的情况下也能够测量气道纤毛细胞中的[Cl]。
