Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sod ium absorption via activation of apical Na/H exchange. HT29-C1 cells were u sed previously to demonstrate that transepithelial SCFA gradients selective ly activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracel lular pH (pH(i)) in HT29-C1 cells, to find out which Na/H exchanger isoform s 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 dos e (ID50) = 1.6 and 0.05 mu M] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (api cal ID50 = 1.4 mu M, basolateral ID50 = 0.3 mu M) RT-PCR detected mRNA of N a/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy o f filter-attached cells reported HOE-694-sensitive pH(i) recovery in respon se to luminal or serosal 130 mM propionate. Confocal analysis along the api cal-to-basal axis revealed that 1) luminal or serosal propionate establishe s transcellular pHi gradients and 2) the predominant site of pHi acidificat ion and pHi recovery is the apical portion of cells. Luminal propionate pro duced 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 apica l NHE2 and dampening of basolateral NHE1 during pi-Ii regulation.