FIG. 1.

V-ATPase-dependent, LY294002 (LY)-sensitive acidification of intracellular compartments of renal epithelial cells in response to glucose deprivation/stimulation. HK-2 cells (a) or LLC-PK₁ cells (a and b) were incubated in standard medium containing serum and glucose on coverslips. Sixteen to eighteen hours prior to the experiment cells were transferred to glucose-free medium containing dialyzed serum and 0.1 mM glucose. Concanamycin A (Con. A) (100 nM), LY294002 (25 μM), and vehicle were added 60 min before stimulation with glucose or d-mannitol (25 mM). Cells were labeled with DAMP, fixed, and stained with fluorescein-conjugated antidinitrophenol antibody. (a) Bright fluorescent structures are acidic compartments. (Upper panels) Acidification of intracellular compartments is induced by glucose but not by the same concentration of d-mannitol. (Lower panels) LY294002 and concanamycin A prevent the effect of glucose. Results shown are representative of five independent experiments. (b) Analysis of fluorescence intensity in LLC-PK₁ cells labeled with DAMP. Image analysis of the average fluorescence intensities in cytoplasmic rectangular areas and fluorescence intensity profiles of cross-sections (see Fig. S3 in the supplemental material) were performed using NIH-Scion Image software. Average fluorescence (mean ± standard error of the mean, arbitrary units [AU]) for 18 to 24 cells from three or four randomly chosen fields is shown.