Cellular SCFA content of colonic epithelial cells (HT29-C1) is dependent on regulation of intracellular and extracellular pH

Abstract

Colonocyte uptake of short-chain fatty acids (SCFAs) is required for physiologic effects of these weak acids on cell physiology and transepithelial ion transport. We have proposed that changes in pH alter SCFA transport, but testing this model requires measurement of intracellular SCFA content, which has not been possible previously. The aim of this study was to use nuclear magnetic resonance (NMR) to directly measure SCFAs, and question whether changes in pH affected intracellular SCFA content. Methods. HT29-C1 cells grown on polystyrene beads were perfused while spectra were acquired on a GE Omega 400 NMR spectrometer. Interleaved non-diffusion weighted (single pulse acquisition) and diffusion weighted 1H NMR spectroscopy (recording the stimulated echo) was used to measure total SCFA, intracellular SCFA and intracellular water volume. SCFA concentration was estimated by integrating the 13-proton signal of iso-butyrate or the sum of the ct, 13, and /-proton peaks of n-butyrate. Correlative experiments used confocal microscopy of cells loaded with SNARF-1 to measure intracellular pH of HT29-C1 cells on beads. Results. NMR spectra were recorded during exposure of cells to SCFA superfusates containing 130mM Na-SCFA substituted mol:mol for NaC1 in isosmotic medium without added bicarbonate/CO s. Concomitant with detection of SCFA in the perfusion buffer, intracellular iso-butyrate or n-butyrate was detected. After extracellular SCFA levels reached steady state, intracellular SCFA content and cell volume continued to slowly increase. We hypothesized that the slow uptake was due to intracellular pH alkalinization and subsequent SCFA redistribution via nonionic diffusion. Amiloride (1 mM) inhibited intracellular SCFA accumulation by 53%, suggesting that the intracellular alkalinization mediated by Na/H exchange was important for regulating cellular SCFA content. Conversely, acidifying perfusates from pH 7.4 to 7.0 stimulated the accumulation of iso-butyrate (35% increase), consistent with increased SCFA uptake by nonionic diffusion. Conclusions. (1) Proton NMR can be used to report on intracellular SCFA content in substrate-attached epithelial cells. (2) SCFA content (and presumably transepithelial SCFA transport) is regulated by pH. This research was funded by the NIH.