Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.
J Neurophysiol. 2012 Feb;107(3):868-79. doi: 10.1152/jn.00878.2011. Epub 2011 Nov 16.
Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.
细胞外的 H(+) 被假设为介导水平细胞对脊椎动物光感受器的反馈抑制。根据这一假说，水平细胞的去极化应该诱导细胞膜附近的细胞外酸化。测试这一假说的实验产生了相互矛盾的结果。研究鲤鱼和金鱼水平细胞的实验，用 pH 敏感染料 5-十六烷酰氨基荧光素 (HAF) 进行了负载，报告说谷氨酸或钾去极化时会发生细胞外酸化。然而，使用 H(+)-选择性微电极的研究报告说，在软骨鱼和鲶鱼水平细胞去极化时会发生细胞外碱化。这些研究在物种、细胞外 pH 缓冲液的使用以及钴的存在与否方面存在差异。我们使用这两种技术在相同的实验条件下（1mMHEPES，无钴）检查来自分离的鲶鱼水平细胞的 H(+) 变化。HAF 荧光表明，高细胞外钾或谷氨酸会引起酸化反应。然而，在相同条件下，使用 H(+)-选择性微电极发现明显的细胞外碱化。共聚焦显微镜显示，HAF 不仅定位于细胞外表面，而且还存在于整个细胞内区室中。HAF 与线粒体特异性染料 MitoTracker 高度共定位。当从细胞中心的光学切片监测 HAF 荧光时，谷氨酸产生细胞内酸化。这些结果与一种模型一致，即去极化允许钙内流，随后激活 Ca(2+)/H(+) 质膜 ATP 酶。我们的结果表明，HAF 报告细胞内 pH 变化，水平细胞的去极化诱导细胞外碱化，这可能缓解 H(+)-介导的光感受器突触传递抑制。
