Role of luminal ATP in the regulation of sodium-hydrogen exchanger 3 (NHE3) trafficking in principal cells (PCs) of cauda epididymis. (A) 3D confocal microscopy reconstruction from the cauda epididymis perfused at the alkaline pH of 7.8 in the absence (left panel) or presence of 100 μm ATPγS (right panel). NHE3 is labeled in red and nuclei are visualized in blue with DAPI. Images were acquired with a 20X/0.8 objective. Each box = 20 µm2. (B) Single confocal images for NHE3 (red) merged with bright-field images from cauda epididymis perfused at pH 7.8 or pH 7.8 + ATPγS (top panels). The white drawing shows the stereocilia area (bottom panels; stereocilia area). The yellow lines show the selected NHE3-associated pixels within stereocilia (bottom panels; NHE3 in stereocilia). Images were taken with a 40X/1.4 objective with a digital zoom of 2.8. Scale bar = 10 µm. (C) A significant decrease in the area occupied by NHE3 in the stereocilia was observed at pH 7.8 + ATPγS when compared to pH 7.8 alone. The values are expressed as percentage of NHE3-associated area versus the stereocilia area. Data are shown as box and whiskers (min to max); n = 5. Each dot represents one epididymis. ***P = .0003 by the Mann-Whitney nonparametric test.
