Regulation of NHE3 subcellular localization in epididymal principal cells: pH, cyclic adenosine 3,5 monophosphate (cAMP), and adenosine signaling

Abstract Introduction The epididymis creates an optimal acidic luminal environment for sperm maturation and storage. In epididymal principal cells (PCs), proton secretion is activated by the accumulation of the sodium‐proton exchanger type 3, NHE3 (SLC9A3), in apical stereocilia. PCs also secrete ATP, which is hydrolyzed into adenosine by ectonucleotidases. Adenosine has opposite effects depending on which purinergic receptors it activates. Activation of ADORA1 (A1) and ADORA3 (A3) receptors decreases intracellular cAMP (cAMP), while activation of ADORA2A (A2A) and ADORA2B (A2B) receptors increases cAMP. In other epithelia, cAMP triggers NHE3 internalization from the apical membrane. Here, we examined the roles of pH, cAMP, and adenosine (via A3, A2A, and A2B receptors) in the subcellular localization of NHE3 in PCs. Methods 3D immunofluorescence confocal microscopy was used to visualize NHE3 in stereocilia or intracellular vesicles. Single confocal microscopy images superimposed with bright‐field imaging was used to quantify NHE3 subcellular localization. The lumen of the cauda (Cd) epididymis of C57Bl/6Ncrl mice was perfused in vivo at pH 6.0 and 7.8. The effect of a permeant analog of cAMP (cpt‐cAMP) was studied at pH 7.8, while the effect of adenosine was investigated at pH 6.0. Expression of A2A, A2B, and A3 was examined by immunofluorescence, and their respective role was evaluated by using specific agonists and antagonists at different luminal pH. Immunofluorescence for clathrin, an endosomal marker, was examined at pH 7.8 with and without an A2B agonist. Results At an acidic pH perfusion solution of 6.0, NHE3 was predominantly localized intracellularly, whereas an alkaline pH of 7.8 promoted its accumulation in apical stereocilia. Perfusion with cpt‐cAMP at pH 7.8 reduced the amount of NHE3 in stereocilia. Immunolabeling showed the localization of A3, A2A, and A2B receptors in the apical membrane of epithelial cells in the Cd epididymis. Adenosine and an A3 agonist increased NHE3 stereocilia accumulation at pH 6.0, and the adenosine effect was abolished with an A3 antagonist. An A2A agonist had no effect on NHE3 localization, while an A2B agonist decreased the amount of NHE3 in stereocilia observed at pH 7.8. A concomitant increase in intracellular labeling for clathrin was induced by the A2B agonist at pH 7.8. Conclusions Our study indicates that in the Cd epididymis, NHE3 localization in PCs is modulated by luminal pH, cAMP, and adenosine receptor signaling. Acidic pH promotes NHE3 internalization, while alkaline pH facilitates its accumulation in stereocilia. Activation of A3 by luminal adenosine maintains NHE3 on the cell surface. Conversely, A2B activation by adenosine induces NHE3 internalization. We propose that the distinct effects mediated by these receptors are the consequence of their opposite effect on cAMP signaling. This intricate interplay of pH and adenosine highlights some of the regulatory mechanisms influencing the establishment of an optimal acidic environment for sperm maturation and storage in the epididymis.