Abstract
The fluoroprobe technique using a potential-sensitive dye, diS-C3(5), was applied to isolated enterocytes in order to correlate membrane potential changes with Na+/sugar cotransport. Cells were prepared from the chicken small intestine by enzymatic (Dispase) treatment combined with mechanical agitation. In the presence of Na+, addition of D-glucose (2mM) to the suspension of cells doped with the dye (1.8μM) gave rise to Na+-dependent increases in fluorescence indicative of depolarization of the cell membrane potential. The pH optimum for this response was ca. 7.3. Similar but smaller fluorescence increases were also evoked by D-galactose, α-methyl-D-glucoside, and 3-O-methyl-D-glucose, all known to be actively accumulated by enterocytes in a Na+-dependent fashion. These monosaccharide-evoked fluorescence changes were suppressed by lowering the extracellular Na+ concentration or by phlorizin. Monosaccharides with no reported Na+ requirement induced no such fluorescence responses. A disaccharide, maltose, elicited a smaller fluorescence increase which also was Na+ dependent and phlorizin inhibitable; this was interpreted as due to a possible supply of D-glucose by the membrane-bound disaccharidase systems. No change was observed with lactose, sucrose, or trehalose, however. The glucose-evoked fluorescence changes correlatable with depolarization were also confirmed using another carbocyanine, diO-C5(3). These results demonstrate that fluorescence from diS-C3(5) or diO-C5(3) can be a good measure for cotransport-associated membrane potential changes in isolated intestinal epithelial cells.
