Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-).
Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-).
复制标题
哺乳动物肾 S3 段的细胞 pH 值和 H( ) 分泌：H( )-ATPase 和 Cl(-) 的作用。
DOI：
10.1007/s002320010019
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Geibel,JP
中科院分区：
文献类型：
--
作者：
Malnic,G;Geibel,JP
应用微灌流技术和共聚焦显微镜研究了H ~+-ATP酶在近曲小管细胞pH调节中的作用。在第一系列实验中，兔肾的近端S3节段被灌注“体外”，而它们的细胞pH值在加载BCECF后通过荧光显微镜测量。在不含Na+和Cl−的培养基中，细胞pH值平均下降0.37 ± 0.051个pH单位，但几分钟后又开始缓慢上升。这种升高为0.17 ± 0.022 pH单位/min，巴弗洛霉素和Cl−通道阻断剂NPPB可显著降低这种升高，但DIDS不能。在第二个系列的实验中，亚细胞囊泡的近端小管细胞的小鼠肾S3段进行了研究，通过共聚焦显微镜后可视化的吖啶橙子或荧光黄。用低Na+溶液灌注后，预期会引起细胞酸化，最初位于基底外侧和核周细胞区域的囊泡向顶端区域移动，如通过NIH Image程序测量的荧光密度变化所检测。在NaCl林格氏液灌注过程中，顶侧与基底侧荧光比率随时间的变化为0.0018 ± 0.0021 min-1，与零无显著差异（P> 0.42）。对于Na+0林格氏液灌流，该变化为0.081 ± 0.015 min-1，P< 0.001。这些斜率被Cl−通道阻断剂NPPB和钒酸盐显著降低，钒酸盐的浓度已被证明会破坏细胞骨架功能。这些数据表明，在无Na+的培养基中，近曲小管细胞的延迟碱化可能是由于液泡H+-ATP酶，其活性在Cl−存在下受到刺激，并依赖于亚细胞囊泡的顶端插入。这些囊泡的运动也依赖于Cl−和细胞骨架的完整性。
The role of H+-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused ``in vitro'' while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na+- and Cl−-free medium, cell pH fell by a mean of 0.37 ± 0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 ± 0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl−channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na+solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018 ± 0.0021 min−1, not significantly different from zero (P> 0.42). For superfusion with Na+0 Ringer, this variation was 0.081 ± 0.015 min−1,P< 0.001 against 0. These slopes were markedly reduced by the Cl−channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na+-free medium is probably due to a vacuolar H+-ATPase, whose activity is stimulated in the presence of Cl−, and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl−and on the integrity of the cytoskeleton.