Gonda T, Maouyo D, Rees S E, Montrose M H
Departments of Medicine and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Am J Physiol. 1999 Jan;276(1):G259-70. doi: 10.1152/ajpgi.1999.276.1.G259.
Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHi gradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 microM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50 = 1.4 microM, basolateral ID50 = 0.3 microM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHi recovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHi gradients and 2) the predominant site of pHi acidification and pHi recovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHi regulation.
结肠腔短链脂肪酸（SCFA）通过激活顶端Na/H交换来刺激电中性钠吸收。先前使用HT29-C1细胞证明跨上皮SCFA梯度可选择性激活极化的Na/H交换体。分别使用荧光测定法和共聚焦显微镜（分别使用BCECF和羧基SNARF-1）来测量HT29-C1细胞内的pH值（pHi），以确定表达了哪些Na/H交换体亚型以及结果是否归因于pHi梯度。HOE-694对Na/H交换的抑制作用确定了：1）悬浮细胞中有两个抑制位点[半数抑制剂量（ID50）= 1.6和0.05微摩尔]；2）滤膜贴壁细胞的顶端和基底外侧膜中各有一个抑制位点（顶端ID50 = 1.4微摩尔，基底外侧ID50 = 0.3微摩尔）。逆转录聚合酶链反应（RT-PCR）检测到Na/H交换体亚型NHE1和NHE2的信使核糖核酸（mRNA），但未检测到NHE3的mRNA。滤膜贴壁细胞的共聚焦显微镜检查显示，响应管腔或浆膜侧130 mM丙酸盐，HOE-694敏感的pHi恢复。沿顶端到基底轴的共聚焦分析显示：1）管腔或浆膜侧丙酸盐建立跨细胞pHi梯度；2）pHi酸化和pHi恢复的主要部位是细胞的顶端部分。与浆膜侧丙酸盐相比，管腔侧丙酸盐使细胞顶端部分的酸化程度明显更高，但未观察到对SCFA梯度方向的其他依赖性。结果为一种亚细胞反应提供了直接证据，该反应可确保在pHi调节过程中顶端NHE2的强劲激活和基底外侧NHE1的抑制。
