pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers

Abstract

In the colonic mucosa, short-chain fatty acids change intracellular pH (pHi) and extracellular pH (pHe). In this report, confocal microscopy and dual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were used for direct evaluation of pHi and pHe in a simple model epithelium, HT29-C1 cells. Live cell imaging along the apical-to-basal axis of filter-grown cells allowed simultaneous measurement of pH in the aqueous environment near the apical membrane, the lateral membrane, and the basal membrane. Subapical cytoplasm reported the largest changes in pHi after isosmotic addition of 130 mM propionate or 30 mM NH4Cl. In resting cells and cells with an imposed acid load, lateral membranes had pHi values intermediate between the relatively acidic subapical region (pH 6.3–6.9) and the relatively alkaline basal pole of the cells (pH 7.4–7.1). Transcellular pHigradients were diminished or eliminated during an induced alkaline load. Propionate differentially altered pHe near the apical membrane, in lateral intracellular spaces between adjacent cells, and near the basal membrane. Luminal or serosal propionate caused alkalinization of the cis compartment (where propionate was added) but acidification of the trans compartment only in response to luminal propionate. Addition of NH4Cl produced qualitatively opposite pHe excursions. The microscopic values of pHi and pHe can explain a portion of the selective activation of polarized Na/H exchangers observed in HT29-C1 cells in the presence of transepithelial propionate gradients.