Unravelling purinergic regulation in the epididymis: activation of V‐ATPase‐dependent acidification by luminal ATP and adenosine

Abstract

Key points In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V‐ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH‐sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V‐ATPase‐dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton‐secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. Abstract Cell–cell cross‐talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V‐ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton‐secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.

Epithelial dynamics in the epididymis: role in the maturation, protection, and storage of spermatozoa

“…In this manner, we found that proton secretion in CCs is activated by an elevation of luminal pH, which induces the trafficking of V‐ATPase‐containing vesicles from the apical pole to the plasma membrane. This results in the formation and elongation of numerous apical projections (Pastor‐Soler et al ., ; Beaulieu et al ., ; Park et al ., ; Battistone et al ., ). Based on their appearance on thin (5 μm) sections used for immunofluorescence labeling, we initially named these protrusions ‘microvilli’ (Breton et al ., ).…”

“…B; green), ‘activated’ CCs perfused at the alkaline pH of 7.8 had numerous microplicae labeled for the V‐ATPase and less intracellular reactivity (Fig. C; green; Pastor‐Soler et al ., ; Beaulieu et al ., ; Park et al ., ; Battistone et al ., ). Addition of bicarbonate, angiotensin II (ANGII), ATP, or adenosine to the luminal perfusate all induced the redistribution of V‐ATPase from intracellular vesicles to the apical membrane (Pastor‐Soler et al ., ; Beaulieu et al ., ; Shum et al ., ; Belleannee et al ., ).…”

“…Alternatively, hydrolysis of ATP to form adenosine could play a role in pH sensing via pH‐dependent ectonucleotidases that are also located on the apical surface of the epididymal epithelium. Indeed, we detected a higher luminal adenosine concentration and a lower ATP concentration when the cauda epididymis was perfused at the alkaline pH of 7.8 vs. pH 6.6 (Battistone et al ., ). This was attributed to activation by alkaline pH of the ectonucleotidases, ecto‐nucleotide pyrophosphatase/phosphodiesterase (ENPP), and ectonucleoside triphosphate diphosphohydrolase (ENTPD), which hydrolyze ATP into ADP, as well as ecto‐5′‐nucleotidase (NT5E), which hydrolyzes AMP to produce adenosine (Yegutkin, ).…”

“…Impairment of purinergic regulation leads to several diseases, including male infertility, and numerous purinergic receptors and ectonucleotidases have been described in the epididymis (Mulryan et al ., ; Robson et al ., ; Martin‐Satue et al ., , ; Banks et al ., ; Belleannee et al ., ; Burnstock, ; Kauffenstein et al ., ; Gorodeski, ). Thus, purinergic signaling would allow CCs to respond to variations in luminal pH in order to maintain an acidic lumen, which as discussed above, is essential for male fertility (Battistone et al ., ). Importantly, our previous studies point toward a role for principal cells (PCs) in ATP secretion and their communication with CCs via cell–cell crosstalk to regulate luminal pH.…”

“…While CBX did not modify the low ATP concentration detected at pH 7.8, it decreased adenosine concentration. Altogether, these results indicated that ATP secretion by PCs, followed by its rapid hydrolysis to produce adenosine, participates in the pH‐induced activation of V‐ATPase‐dependent proton secretion by CCs (Battistone et al ., ). In this model, CFTR is a key player in the PC–CC crosstalk as it mediates both bicarbonate and ATP transport.…”