Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis

An acidic luminal pH in the epididymis and vas deferens (VD) helps maintain mature sperm in an immotile state during storage. We have previously shown that the majority of proton secretion in the VD is due to the activity of the vacuolar H+-ATPase. Acidification is dependent on luminal sodium in mor e proximal regions of the epididymis, and we examined the distribution of t he Na+/H+ exchanger, NHE3, by immunofluorescence and measured Na+/H+ exchan ge (NHE) activity in isolated epididymal tubules. NHE3 was detected in the apical pole of nonciliated cells of the efferent ducts and principal cells (PC) of the epididymis. No staining was seen in the distal cauda epididymid is and the VD. Isolated tubules from the distal initial segment (DIS) and p roximal cauda epididymidis were perfused in vitro and loaded with the pH-se nsitive dye 2',7'-bis(carboxyethyl)5(6')-carboxyfluorescein. Ethylisopropyl amiloride (EIPA) (50 muM) reduced the initial rate of intracellular pH rec overy (dpH(i) dt), in response to an acute acid load, by 51% and 45% in the DIS and cauda epididymidis, respectively. In the DIS, removal of luminal s odium reduced dpH(i)/dt by 52%. HOE694 (50 mM) inhibited all EIPA-sensitive dpH(i)/dt in the DIS, despite the previously reported absence of NHE2 in t his region (Cheng Chew SB, Leung GPH, Leung PY, Tse CM, and Wong PYD, Biol Reprod 62: 755-758, 2000). These data indicate that HOE694- and EIPA-sensit ive Na+/H+ exchange may participate, together with the H+-ATPase, in lumina l acidification in the male excurrent duct.