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 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.
Gonda
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Departments of Medicine and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Maouyo
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Rees
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Montrose
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D001578:Benzopyrans; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects; D005232:Fatty Acids, Volatile / Q000008:administration & dosage / Q000494:pharmacology / Q000502:physiology*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007536:Isomerism; D018613:Microscopy, Confocal; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism / Q000502:physiology*; D014407:Tumor Cells, Cultured
D001578:Benzopyrans; D005232:Fatty Acids, Volatile; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; C068301:carboxy-seminaphthorhodaminefluoride; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein