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 (pH(i)) of isolated colonocytes was affected by exposure to 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 iso-butyrate in isosmotic Na+-free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pH(i) alkalinization (Na+-independent pH(i) recovery). Upon subsequent removal of the SCFA, pH(i) alkalinized beyond the starting pH(i) (a pH(i) overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pH(i) 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 pH(i) alkalinization. The Na+-independent pH(i) 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, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), α- 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 pH(i) recovery.