Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, USA.
ACS Chem Biol. 2013 Mar 15;8(3):636-42. doi: 10.1021/cb300396j. Epub 2013 Jan 7.
Unlike the digestive systems of vertebrate animals, the lumen of the alimentary canal of Caenorhabditis elegans is unsegmented and weakly acidic (pH ~4.4), with ultradian fluctuations to pH > 6 every 45-50 s. To probe the dynamics of this acidity, we synthesized novel acid-activated fluorophores termed Kansas Reds. These dicationic derivatives of rhodamine B become concentrated in the lumen of the intestine of living C. elegans and exhibit tunable pKa values (2.3-5.4), controlled by the extent of fluorination of an alkylamine substituent, that allow imaging of a range of acidic fluids in vivo. Fluorescence video microscopy of animals freely feeding on these fluorophores revealed that acidity in the C. elegans intestine is discontinuous; the posterior intestine contains a large acidic segment flanked by a smaller region of higher pH at the posterior-most end. Remarkably, during the defecation motor program, this hot spot of acidity rapidly moves from the posterior intestine to the anterior-most intestine where it becomes localized for up to 7 s every 45-50 s. Studies of pH-insensitive and base-activated fluorophores as well as mutant and transgenic animals revealed that this dynamic wave of acidity requires the proton exchanger PBO-4, does not involve substantial movement of fluid, and likely involves the sequential activation of proton transporters on the apical surface of intestinal cells. Lacking a specific organ that sequesters low pH, C. elegans compartmentalizes acidity by producing of a dynamic hot spot of protons that rhythmically migrates from the posterior to anterior intestine.
与脊椎动物的消化系统不同，秀丽隐杆线虫的消化道管腔未分段且呈弱酸性（pH 值约为 4.4），每隔 45-50 秒就会出现 pH 值超过 6 的超低频波动。为了探究这种酸度的动态变化，我们合成了一种新型的酸激活荧光染料，称为堪萨斯红。这些罗丹明 B 的二价阳离子衍生物在活体秀丽隐杆线虫的肠道管腔中浓缩，并表现出可调的 pKa 值（2.3-5.4），受烷基胺取代基的氟化程度控制，允许在体内成像一系列酸性流体。对自由摄取这些荧光染料的动物进行荧光视频显微镜观察显示，秀丽隐杆线虫肠道中的酸度是不连续的；后肠包含一个大的酸性段，其两侧是后肠末端较小的较高 pH 值区域。值得注意的是，在排粪运动程序期间，这个酸度热点迅速从前肠向后肠移动，在前肠中每 45-50 秒定位长达 7 秒。对 pH 值不敏感和碱基激活荧光染料以及突变体和转基因动物的研究表明，这种动态酸度波需要质子交换器 PBO-4，不涉及流体的大量移动，并且可能涉及肠细胞顶表面上质子转运体的顺序激活。由于缺乏隔离低 pH 值的特定器官，秀丽隐杆线虫通过产生质子的动态热点来分隔酸度，该热点从后肠到前肠呈节律性迁移。
