An Na(+)-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes.

Abstract

Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure m SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or /s0-butyrate in isosmotic Na+-free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na § pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a phi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and phi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na+-independent phi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K + gradient, CI-gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), ~-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na+-independent pHi recovery.

Intracellular pH regulation in guinea-pig caecal and colonic enterocytes during and after loading with short-chain fatty acids and ammonia

“…Decreasing the pH from 7.4 to 6.7 increases the concentration of the undissociated butyric acid 5-fold from 0.29 to 1.46 mmol/l. Diffusion of SCFA in the protonated form across hind gut epithelium has been emphasized in various recent studies [6,7,37]. Similar findings have been reported for crypt cells from the distal colon of mice [9] and in isolated perfused crypts in mice [38] and rats [14].…”

“…Intracellular pH (pH i ) regulation in epithelial cells of the large intestine is therefore of considerable interest. Recent studies have focused on effects of SCFA on the pH i of colonic tumour cells [13,22,35] or on isolated colonic crypts [7,14], but only limited information is available on the regulation of pH i in intact colonic epithelial sheets [8,11,18,37]. With respect to the location of pH i -regulating mechanisms these studies are somewhat contradictory and interpretation has been complicated further by recent observations in perfused crypts suggesting that the apical membranes of the colonic and gastric epithelia are less permeable for lipid soluble substances, such as SCFA and NH 3 , than the respective basolateral membranes [14,38,47].…”

Role of short‐chain fatty acids in the hind gut

“…The pH in the luminal microclimate region is close to neutral and largely independent of changes in bulk luminal pH; The pH in the microclimate depends substantially on the presence of bicarbonate (42). Addition of SCFA to isolated colonocytes acidified the intracellular pH (17,24). However, addition of SCFA to the luminal side of the in vitro isolated intact caecal or distal colonic epithelia of guinea pig or to monolayers of HT29 cells had no major effect on pH1, independently whether the apical Na+-H+ exchange or the apical K+-H+ ATPase had been inhibited or not (12).…”

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

“…SCFAs (ie. propionate, butyrate, acetate) are commonly present in the lumen of the large intestine at 100-150 mM total concentration [35], ammonium is present at 20-30 mM [37], and sodium is 10-20 mM [38][39][40]. Our results suggest that all these unusual conditions could potentially modify activation of one or more apical NHEs by translocation of the protein to the membrane.…”

“…8B and 8C) was not significantly different (P>0.05) in magnitude from the pH i decrease caused by ammonium exposure. In Figures 8B and 8C, Na + -dependent pH recovery was not evident in the continued presence of the weak acid, propionate, a condition which acts to keep pH i clamped at a low level [35]. We conclude that in the absence of external sodium, acidification by propionate or ammonium was equally effective at recruiting both NHE2-CFP and NHE3-CFP to the PM.…”